A biomarker is a biological characteristic that is measured and evaluated objectively as an indicator of normal biologic processes, pathogenic processes, or pharmacologic response to therapeutic intervention (Hewitt et al., 2004) The identification of clinically useful, validated, protein biomarkers in biological fluids is a major goal for proteomics as this would allow for the earlier detection and reclassification of a wide range of pathological disorders. The proteome of body fluids is complex (Anderson and Anderson, 2002) and proteomics on these fluids is challenging as the more abundant proteins, for example albumin in plasma and Tamm-Horsfall Protein (THP) in urine, are more likely to be detected and the less abundant, and more interesting and informative proteins are difficult to identify.
Multiple organ injury has high mortality and there is a need to understand the pathological nature of how the organ injury arises. Drug overdose (for example paracetamol overdose) can induce multiple organ failure and a key mediator of paracetamol induced organ injury is the transcription factor c-Jun.
Deliberate or accidental overdose with paracetamol (also known as acetoaminophen—APAP) is the most common cause of poisoning in the United Kingdom. For example, around 1 person per day requires admission to the Royal Infirmary of Edinburgh for treatment of a significant APAP overdose (unpublished data). However, only around 12% of admissions will develop any degree of organ injury and only around 50 patients per year from all of Scotland are referred to the Scottish Liver Transplant Unit with severe APAP—induced liver injury (unpublished data). APAP overdose is treated with a glutathione pro-drug (N-acetylcysteine (NAC)) but adverse reactions to this antidote are common (around 15% of patients). As the majority of patients will not develop organ injury we may be over-treating with a potentially toxic antidote.
The need for NAC treatment is determined by the blood paracetamol concentration. Those found to have a sufficiently high concentration of paracetamol in their blood are administered NAC intravenously over a period of approximately 20 hours. Once the NAC treatment is complete, further tests to assess the extent of any liver damage are conducted. These tests require the measurement of serum transaminase enzymes (ALT and AST), blood clotting (INR) and serum creatinine. As such, paracetamol induced organ damage may, at present, only be excluded after a minimum period of 20 hours.
In addition, adverse reaction to NAC are common and as such it is desirable to reduce the length of NAC infusion.
Existing biomarkers of APAP-induced organ injury (for example serum alanine transaminase (ALT), international normalized ratio (INR) and creatinine) do not accurately detect or exclude injury until around 24 hours post-APAP ingestion. If we could detect or exclude injury earlier then we could reduce the number and/or length of hospital admissions.
The ability of eukaryotic cells to release components of their cell membrane was first described by Wolf in 1967 and eloquently termed ‘platelet dust’. Essentially all cell types release small membrane vesicles, today known as microparticles or exosomes, especially when the cells are activated or during apoptosis (George et al., 1982).
The urinary proteome is easier to investigate than blood and large amounts can be collected. All nephron segments of the kidney normally secrete small vesicles containing apical membranes and intracellular fluid into the urine and these vesicles, termed exosomes, are less than 80 nm in size (Johnstone et al., 1987). Exosomes are orientated ‘cytoplasmic-side inward’ which is a unique characteristic of these vesicles. Exosomes can be isolated from urine by differential centrifugation and several renal disease-related proteins have been identified in these exosomes through liquid-chromatography-tandem mass spectrometry (LC-MS/MS) (Pisitkun et al., 2004). Zhou et al. (2006) established the optimal conditions for the collection and storage of urine exosomes to maximise the amount of exosomal protein that can be isolated and, therefore, facilitate protein identification. Urinary exosomes could provide key biomarkers of renal disease as their numbers and composition could be altered during disease and may give an indication of organ dysfunction. It should be noted, however, that urinary exosomes might reflect changes in circulating blood MPs as well as changes in kidney function. Therefore, urinary exosomal protein may represent a source of biomarkers for systemic disease. The ability to isolate and characterise urine exosomes may be the first step in identifying disease biomarkers.
It is among the objects of the present invention to obviate or mitigate the abovementioned problems with the prior art.